A crucial step in the emergence of new waterborne and foodborne pathogens in developed countries is the evolution of durability traits that allow bacteria to survive in mass water and food distribution systems. For Escherichia coli O157:H7, a foodborne pathogen and growing public health problem, the ability to tolerate extreme acidity enables this organism to endure in foods and infect susceptible human in very low doses. In the research proposed here, the role of natural selection for acid tolerance and other aspects of durability in the external environment will be tested in an experimental system of bacterial evolution. The design simulates the transition between the two principal habitats of an enteric pathogen; the primary habitat in the intestine and the secondary habitat in the water, soil, and food. The conditions of the secondary environment will be varied to impart different selection pressures against which pathogenic strains can evolve. The environments range from relatively benign favoring growth, to a harsh environment favoring improved durability. The specific aims are: (1) to experimentally determine how variables which influence survival in the external environment affect the direction and rate of evolution of durability; (2) to investigate the evolution of the components of durability (acid, salt, and heat tolerance, resistance to desiccation) and their correlated change with selection for single resistance factors; (3) to quantify variation in durability among E. coli from natural environments and assess the potential for emergence of new pathogenic strains; and (4) to elucidate the genetic basis of evolutionary change in durability by characterizing mutations in genes known to influence acid tolerance and other protection mechanisms. The evolved strains will be compared to the ancestral (original) strains in their durability and the costs (in terms of reduced fitness) of adaptation to the secondary environment will be assessed. In addition, the durability of E. coli from natural environments and outbreaks of disease will be compared to the evolved levels of durability. The molecular basis of evolved durability will be investigated through subtractive hybridization to identify genes that contribute to protection and cross protection against environmental challenges.